Method of determining unsaturated iron binding capacity in serum

ABSTRACT

Unsaturated iron binding capacity of transferrin, a serum protein, is determined by adding an iron solution containing a known quantity of excess iron to the serum to saturate the iron binding capacity of the serum transferrin, removing the remaining surplus iron with an absorbent and making a colorimetric determination by causing iron to be released from transferrin under an acidic condition. Since the iron solution is unstable in alkaline zone, it is stabilized by forming it into nitrilotriacetic acid iron chelates. The stability of iron chelates of sodium bathophenanthroline sulfonate is greater than that of nitrilotriacetic acid. This fact is utilized, and the surplus quantity of nitrilotriacetic acid chelates remaining after the saturation of the iron binding capacity of tranferrin is measured by using sodium bathphenanthroline sulfonate. The sodium bathophenanthroline sulfonate iron chelates have maximum absorbance at the wavelength of 535 nm, and Beer&#39;&#39;s law holds for an iron concentration range up to 600 mg/100 ml.

Unite States atent [1 1 Takase et al.

[ METHOD OF DETERMINING UNSATURATED IRON BINDING CAPACITY IN SERUM [75]Inventors: Kazuei Takase; Junji Morikawa,

both of Tokyo, Japan {73] ,Assignee: Eiken Chemical Co., Ltd., Tokyo,

Japan [22] Filed: Apr. 5, 1973 [21] ,Appl. No; 348,097

OTHER PUBLICATIONS R, J. Henry, Clinical Chemistry Principles andTechnics, Harper & Row, 1964, pp. 391-395 relied on, RB40.H4.

G. W. Bates, Dissert. Abstr. 27B (ll), 3816 (1967). Bates et al., Chem.Abstr. 67, 7925x (1967).

R. L. Searcy, Diagnostic Chemistry, McGraw Hill, 1969, p. 329 relied on,RB40.54.

Frankel et al, ed, Gradwohls Clinical Laboratory [451 June 3,1975

Methods and Diagnosis, C. V. Mosby C0., 1970. Vol. I, pp. 205-368, 441relied on, RB37.G7.

Primary ExaminerRobert M. Reese Attorney, Agent, or Firm-Stevens, Davis,Miller & Mosher [5 7] ABSTRACT Unsaturated iron binding capacity oftransferrin, a serum protein, is determined by adding an iron solutioncontaining a known quantity of excess iron to the serum to saturate theiron binding capacity of the serum transferrin, removing the remainingsurplus iron with an absorbent and making a colorimetric determinationby causing iron to be released from transferrin under an acidiccondition. Since the iron solution is unstable in alkaline zone, it isstabilized by forming it into nitrilotriacetic acid iron chelates. Thestability of iron chelates of sodium bathophenanthroline sulfonate isgreater than that of nitrilotriacetic acid. This fact is utilized, andthe surplus quantity of nitrilotriacetic acid chelates remaining afterthe saturation of the iron binding capacity of tranferrin is measured byusing sodium bathphenanthroline sulfonate. The sodiumbathophenanthroline sulfonate iron chelates have maximum absorbance atthe wavelength of 535 nm, and Beers law holds for an iron concentrationrange up to 600 mg/lOO ml.

4 Claims, 4 Drawing Figures Absorption Spectra of Fe(II)-BPT ComplexSample serum,

tilisoronnce will! MOO 450 Standard iron so|ution,(2.5 .|g)

Serum blank.

Wpve Length,

Reference.- water MFHTE JU? 3 "975 SHEET Mbsorpflon Spectra ofFe(II)-BPT Complex \PH: 8.6

Reference: wcl'rer \Effect of pH on serum UIBC value ll (PH) \M-Tris.HCLBuffer 'Y Buffer rgwgr-nrmmws 191s u 15% 8 87 332 Relationship of Rateof Color Development to time and Temperature.

M15 20 Standard Iron Solution,

D4 Somp|e Serym Serum B a 5 Mb 1'5 2'0 2'5 3'0 35 (min) Reference WaterFe pg/IOOml METHOD OF DETERMINING UNSATURATED IRON BINDING CAPACITY INSERUM The present invention relates to a method of determining theunsaturated iron binding capacity in serum and, more particularly, to amethod of readily and exactly determining the unsaturated iron bindingcapacity of the serum transferrin.

The clinical significance of blood iron was made clear by Heilmeyer (in1937). Of the total quantity of iron contained in the adult body, 65percent is present in red corpuscles as hemoglobin component (redcorpuscle iron I), and about 30 percent is stored in liver, pancreas,marrow of bones and kidney (stored iron). While iron contained in blood{blood iron) is very slight in quantity, no greater than mere 0.1percent (3 to 5 milligrams), the quantity of iron getting into and outof blood through this blood iron is as great as 30 milligrams or moreper day. This to milligrams of iron is utilized for the production ofhemoglobin in the marrow of bones, so that it may be said that the bloodiron maintains the blood producing function of the marrow.

In the serum the blood iron is bound to transferrin which belongs to,B-globulin. its normal content is 90 to I50 mg/100 ml for men and 80 to120 mg/lOO ml for women. The serum is capable of binding iron up to 300to 360 mg/100 ml. This total capacity is termed total iron bindingcapacity, and the difference between the total iron binding capacity andthe actual blood iron content is termed unsaturated iron bindingcapacity.

The determination of the total iron binding capacity and unsaturatediron binding capacity is utilized in the classification of variousamenic diseases and in the determination and diagnoses of liver diseasesand acute and chronical infectious diseases.

If the determination of the total iron binding capacity and unsaturatediron binding capacity is done along with the determination of thecontent of the blood iron, the diagnoses of various diseases stemmingfrom abnormal iron metabolism can be done more precisely.

Major prior-art methods of determining the unsaturated iron bindingcapacity are as follows;

1. Magnesium carbonate method This method consists of adding an excessiron solution, for instance a ferric chloride solution, to the serum tosaturate the iron binding capacity of transferrin in the serum,subsequently removing surplus iron with magnesium carbonate, removingprotein, thereafter estimating total iron binding capacity through acolorimetric process, and then subtracting the separately determinediron content value from the total iron binding capacity value, therebydetermining the unsaturated iron binding capacity.

2. Peters method This method consists of adding a ferric ammoniumcyanate solution to the serum to saturate the iron binding capacity ofthe serum transferrin, then removing the surplus iron with acation-exchange resin, and after the removal of protein determining theunsaturated iron binding capacity in the same way as mentioned above.

3. RI method This method consists of adding a predetermined quantity ofexcess radioactive isotope Fe59 and iron to the serum to saturate theiron binding capacity of the serum transferrin, causing the residualsurplus iron to be absorbed in a resin sponge and measuring theradioactivity of the absorbed iron.

Of the above prior-art methods, the first two involve many operationsteps and are very tedious. The last method requires special equipmentand uses expensive reagents, so that it is difficult to be carried outin the usual examination room.

To solve the above problems encountered in the prior-art methods, it hasbeen proposed to directly determine the unsaturated iron bindingcapacity by using a tris hydrochloric acid buffer (1.0 M, pH being 8.1),a standard iron solution (prepared by using ferrous ammonium sulfate andcontaining 500 mg/ ml of iron and also used by adding 0.5 mg/lOO ml ofascorbic acid) and 0.5 percent of a bathophenanthroline sulfonatesolution. However, the above salt of iron is unstable and formshydroxides of iron in alkaline liquid. Therefore, it is dissolved in anacidic solution, to which an alkaline buffer is subsequently added forcolor. development and colorimetric determination, so that operation isinvolved.

An object of the present invention is to provide a method of speedilydetermining the unsaturated iron binding capacity of transferrin with areduced number of operational steps.

Another object of the invention is to provide a method of determiningthe unsaturated iron binding capacity of transferrin with high accuracyand which enables one to obtain satisfactory reproducibility.

A further object of the invention is to provide a reagent (II)-BPT inthe determination of unsaturated iron binding capacity of transferrinand one capable of perservation for a long time.

The above and other objects, features and advantages of the inventionwill appear more fully from the following description.

FIG. 1 is a graph showing an absorptive spectral of Fe (II)- BPTcomplex,

FIG. 2 is a graph showing the effect of pH on unsaturated iron bindingcapacity value,

FIG. 3 is a graph showing a relationship of rate of color development totime and temperature, and

FIG. 4 is a graph showing the calibrating curves representing the effectof Fe buffer solutions.

According to this invention there is provided a method of determiningunsaturated iron binding capacity of transferrin by adding an ironsolution containing a known quantity of excess iron to the serum tosaturate the iron binding capacity of the serum transferrin andmeasuring the remaining surplus iron.

To carry out the above method, the problem has been studied of whetheror not the number of operation steps may be reduced by adding a standardiron solution to a tris hydrochloric acid buffer. It has been found thatit is best to establish the standard iron solution in the form of ironchelate compound in order to preserve the solution in the trishydrochloric acid buffer for a long time.

As the chelating agent there may be used ethylenediamine tetraaceticacid (EDTA), bathophenanthroline sulfonate (BPT), tripyridyl-s-triadine(TPTZ) and nitrilotriacetic aicd (NTA). These substances can bind ironto form the so-called iron chelate compound, but their force of bindingiron is different in the following way;

BPT EDTA NTA TPTZ It constitutes the principle underlying the inventionthat the unsaturated iron binding capacity of serum is determined bypreparing a standard solution by dissolving an iron chelate compound inwater, adding a predetermined quantity of sample serum to the standardsolution and measuring the difference of absorbance before and after theaddition of the serum.

More particularly, according to the invention a tris hydrochloric acidbuffer containing the iron chelate compound is added to the serum tosaturate the iron binding capacity of the serum transferrin, the surplusiron not bound to transferrin is removed, ascorbic acid is added,followed by the addition of bathophenanthroline sulfonate forcolorimetric determination.

At this time, if the iron chelate compound is based on BPT or EDTA, thetransferrin in the serum cannot derive iron from the chelates since thebond in the chelates is too strong. On the other hand, NTA and TPTZ haveless strong iron binding forces than those of BPT and EDTA, so thattransferrin can take up a requisite quantity of iron from the ironchelate compound based on NTA or TPTZ. TPTZ-Fe compound, however, isunstable in the tris hydrochloric acid buffer, so that it not suited forpractical use. From these grounds, it has been recognized that theNTA-Fe compound is suitable for use.

Considering another aspect, the unsaturated iron binding capacity ofserum differs with pH. To examine the effect of pH the unsaturated ironbinding capacity has been measured of transferrin in the same serum withthe pH adjusted to various values by using M-tris hydrochloric acidbuffer for a pH range between 7 and 9 and M-glycine sodium hydroxidebuffer for a pH range between 9 and l l. The conclusion is reached thata highly unsaturated iron binding capacity can be maintained within a pHrange between 8.4 and 8.7, as will be described hereinafter inconnection with FIG. 2.

EXAMPLE Preparation of reagents Nitrilotriacetic acid iron buffer:

121.14 grams of tris (hydroxymethyl) aminomethane was dissolved in 800millilitres of distilled water, and 0.08 gram of nitrilotriacetic acidand then 0.01 17 gram of ferrous ammonium sulfate were added to anddissolved in the resultant solution. Then, the pH of the obtainedsolution was adjusted with 6N-hydrochloric acid. Then, distilled waterwas added to the resultant solution to adjust the total Volume to 1,000milliliters. The concentration of iron in the final solution is166.7mg/100 ml.

Nitrilotriacetic acid buffer:

It was prepared in the same way as the aforementioned and has the samecomposition as the aforementioned nitriloacetic acid iron buffer exceptfor the fact that ferrous ammonium sulfate was not added.

Bathophenanthroline sulfonate solution:

It was prepared by dissolving 0.5 gram of sodium bathophenanthrolinesulfonate in distilled water such that the total volume was 100millilitres.

Reducing agent:

A small quantity of sodium ascorbate was used as the reducing agent.

The apparatus used for the preparation of the reagents and for theoperation were all deironated.

Operation Four test tubes A, B, C and D were prepared. 1.5 millilitresof nitrilotriacetic acid iron bufferwas poured into each of the testtubes A, B and C, while 1.5 millilitres of nitriloacetic acid buffer waspoured into the test tube D. Then, 10 milligrams of the reducing agentwas poured into each test tube to produce each blend solution. Then, 0.5millilitre of sample serum was poured into each of sample serum testtube A and serum blank test tube B, while 05 millilitre of distilledwater was poured into each of standard iron solution test tube C andreagent blank test tube D. After leaving these tubes for 10 minutes,0.05 millilitre of sodium bathophenanthroline sulfonate solution waspoured into each of the tubes A, C and D, while 0.05 millilitre ofdistilled water was poured into the remaining tube B. After sufficientlystirring the individual resultant liquids and then leaving then at roomtemperature for 20 minutes, the absorbance of the samples, in theindividual test tubes at the wavelength of 535 nm was measured with aspectrophotometer by using water as a reference.

By so doing, the unsaturated iron binding capacity (UIBC) of the serumcan be determined from an equation A 1553:" UIBC ug/ml) 500 500 where EE E and E are values of the absorbance of the samples in the respectivetest tubes. Absorbance curve Sodium bathophenanthroline sulfonate ironchelates develop red color in alkaline solution, and FIG. 1 shows theresultant absorbance spectra in a wavelength range between 400 and 600nm. Maximum absorbance was recognized at the wavelength of 535 nm. Inthe subsequent experiments the determination of the absorbance was madeonly for this wavelength. Effect of pH on unsaturated iron bindingcapacity value of the serum FIG. 2 shows the results of measurements ofthe unsaturated iron binding capacity value using M-tris hydrocloricacid buffer for a pH range between 7 and 9 and M-glycine sodiumhydroxide buffer for a pH range between 9 and l 1. As is shown, asubstantially constant maximum value was obtained within a pH rangebetween 8.4 and 8.7. Relationship of rate of color development to timeand temperature As shown in FIG. 3, at temperatures about roomtemperature (20C) the absorbance of both sample serum and standard ironsolution settled to respective steady values in about 5 minutes. Attemperatures of 15C the time required until the settling was extended,particularly for the sample serum is about 15 minutes. Effect of timefor the action of serum on nitrilotriacetic acid iron buffer and sodiumascorbate on the unsaturated iron binding capacity value Time until theserum transferrin was saturated with iron by deriving and binding withiron from the nitrilotriacetic acid iron buffer was determined. Theunsaturated iron binding capacity was settled to a constant value afterthe sample had been left for 5 minutes.

Relationship between absorbance and Fe concentration in nitrilotriaceticacid iron buffer A calibration curve was produced with the final Fecontent adjusted to 0, 100, .200, 300, 400 and 500mg/l00 ml for eachnitrilotriacetic acid iron buffer and by using reagent blank asreference. The curve obtained was straight up to 500mg/ l00 ml andpassed the origin, as shown at plot 1 in FIG. 4. Also, the absorbancewas measured in the same way up to the Fe contact of .500mg/100 ml inthe nitrilotriacetic acid iron buffer but using a nitrilotriacetic acidbuffer containing serum as reference. In this case, a straight plotparallel to plot 1 but lower than it by the absorbance valuecorresponding to the unsaturated iron binding capacity of the serum (inthis case 220mg/100 ml) was obtained. as shown at plot 2. It is to benoted that this plot shows absorbance of 0.005 to 0.01 for Fe contentsof 100 and 200mg/100 ml. This is due to the pink coloring of the iron inthe transferrin. This is re-confirmed from the fact that the absorbanceof plot 3 obtained in case of adding only nitrilotriacetic acid ironbuffer and reducing agent but not adding sodium bathophenanthrolinesulfonate to the serum increases linearly from zero Fe content to thesaturation point of 220g/100 m1, and then it remains unchanged forgreater Fe contents. Results of determination of unsaturated ironbinding capacity value in human serum Unsaturatd iron binding capacityvalue for 32 human serum samples was determined by the method accordingto the invention and also through calculation based on the Peter methodand magnesium carbonate method. Table (1) lists the analytical results.Excellent results were found to be obtained by the method according tothe invention, with the co-relation coefficient and regression line withrespect to the value (y) according to the Peter method beingrespectively 0.98 and (y) 1.1.97 10.5 and those with respect to thevalue 1v) according to the magnesium carbonate method being respectively0.90 and (y) 0.98 10.4

the recovery of iron by adding 10, 30 and mg/100 ml of Fe(ll) to eachsample. Table (11) shows the results of the experiments. The recoverywas 89.2 to 103.7 percent and 94.4 percent in the average.

Table (II) Recovery of iron added to serum Sample Fe added Fe foundRecovery No. (pg/ml) (pg/100ml) (pg/100ml) 30 As has been described inthe foregoing, the method according to the invention permits a simpleand speedy operation. Also, according to the invention it is possible toobtain satisfactory precision of determination and recovery. Further,the correlation with the priorart methods using iron absorbants issubstantially satisfactory. Thus, the method according to the inventionis thought to be suitable as practical analytic method for thedetermination of the unsaturated iron binding capacity in serum.

Table 11) t tnalytical results of UIBC value in human serum lug/ 100ml)Sample Proposed Peters MgCQ, Sample Proposed Peters MgCO Nummethodimethod method Nummethod method method ber ber Reproducibility What weclaim is:

The same human serum sample (with unsaturated iron binding capacityvalue of 1.89mg/l00 ml) was an- I. A method of determining theunsaturated iron binding capacity of transferrin in serum comprising thealyzed ten times, and the variation factor was found to 65 steps of:

be 1.3 percent. Recovery Two human serum samples were used experimentingpreparing a standard solution of iron chelate in which a known amount ofiron is bound with nitrilotriacetic acid by using a buffer solution ofpH 8.4 to 8.6;

adding the said standard iron solution to a sample serum in order tosaturate the iron binding capacity of transferrin in the said serum;

reducing the excessive iron remaining unbound with transferrin by addinga salt of ascorbic acid to the said saturated solution;

developing a color by adding sodium bathophenanthroline to the saidreduced solution, and determining the absorbance of the said coloredsolution.

2. The method as defined in claim 1, wherein said standard solutioncontains a small quantity of sodium ascorbate to serve as a reducingagent.

3. A method of determining the unsaturated iron binding capacity ofserum comprising the steps of: pre paring four test tubes; preparing asample serum by pouring 1.5 millilitres of a nitrilotriacetic acidbuffer, 10 milligrams of a reducing agent, 0.5 millilitre of the serumand 0.05 millilitre of sodium bathophenanthro line sulfonate into afirst one of said test tubes; preparing a serum blank by pouring 1.5millilitres of a nitrilotriacetic acid iron buffer, 10 milligrams ofsaid reducing agent, 0.5 millilitre of said serum and 0.05 millilitre ofdistilled water into a second one of said test tubes; preparing astandard iron solution by pouring 1.5 millilitres of saidnitrilotriacetic acid iron buffer, 10 milligrams of said reducing agent,0.5 millilitre of distilled water and 0.05 millilitre of a sodiumbathophenanthroline sulfonate solution into a third one of said testtubes; preparing a reagent blank by pouring 1.5 millilitres of saidnitrilotriacetic acid buffer, 10 milligrams of said reducing agent, 0.5millilitre of distilled water and 0.05 millilitre of said sodiumbathophenanthroline sulfonate solution into the last one of said testtubes; determining the absorbance at the Wavelength of 535 nm of therespective samples in said test tubes by using water as reference; andcalculating the value of the unsaturated iron binding capacity from thevalues of the absorbance of said respective sample.

4. A composition for determining the unsaturated iron binding capacityin serum comprising in proportion 1.5 millilitres of a nitrilotriaceticacid iron buffer, 10 milligrams of a salt of ascorbic acid, 0.5millilitres of distilled Water and 0.05 millilitres of a sodiumbathophenanthroline sulfonate solution.

1. A METHOD OF DETERMINING THE UNSATURATED IRON BINDING CAPACITY OFTRANSFERRIN IN SERUM COMPRISING THE STEP OF: PREPARING A STANDARDSOLUTON OF IRON CHELATE IN WHICH A KNOWNAMOUNT OF IRON IS BOUND WITHNITRILOTRIACETIC ACID BY USING A BUFFER SOLUTION OF PH 8.4 TO 8.6;ADDIBG THE SAID STANDARD IRON SOLUTION TO A SAMPLE SERUM IN ORDER TOSATURATE THE IRON BINDING CAPACITY OF TRANSFERRIN IN THE SAID SERUM; 1.A method of determining the unsaturated iron binding capacity oftransferrin in serum comprising the steps of: preparing a standardsolution of iron chelate in which a known amount of iron is bound withnitrilotriacetic acid by using a buffer solution of pH 8.4 to 8.6;adding the said standard iron solution to a sample serum in order tosaturate the iron binding capacity of transferrin in the said serum;reducing the excessive iron remaining unbound with transferrin by addinga salt of ascorbic acid to the said saturated solution; developing acolor by adding sodium bathophenanthroline to the said reduced solution,and determining the absorbance of the said colored solution.
 2. Themethod as defined in claim 1, wherein said standard solution contains asmall quantity of sodium ascorbate to serve as a reducing agent.
 3. Amethod of determining the unsaturated iron binding capacity of serumcomprising the steps of: preparing four test tubes; preparing a sampleserum by pouring 1.5 millilitres of a nitrilotriacetic acid buffer, 10milligrams of a reducing agent, 0.5 millilitre of the serum and 0.05millilitre of sodium bathophenanthroline sulfonate into a first one ofsaid test tubes; preparing a serum blank by pouring 1.5 millilitres of anitrilotriacetic acid iron buffer, 10 milligrams of said reducing agent,0.5 millilitre of said serum and 0.05 millilitre of distilled water intoa second one of said test tubes; preparing a standard iron solution bypouring 1.5 millilitres of said nitrilotriacetic acid iron buffer, 10milligrams of said reducing agent, 0.5 millilitre of distilled water and0.05 millilitre of a sodium bathophenanthroline sulfonate solution intoa third one of said test tubes; preparing a reagent blank by pouring 1.5millilitres of said nitrilotriacetic acid buffer, 10 milligrams of saidreducing agent, 0.5 millilitre of distilled water and 0.05 millilitre ofsaid sodium bathophenanthroline sulfonate solution into the last one ofsaid test tubes; determining the absorbance at the wavelength of 535 nmof the respective samples in said test tubes by using water asreference; and calculating the value of the unsaturated iron bindingcapacity from the values of the absorbance of said respective sample.